WO2023192433A1 - Procédés et systèmes rapides à usage unique pour l'analyse électrochimique d'agents pathogènes dans l'air expiré - Google Patents

Procédés et systèmes rapides à usage unique pour l'analyse électrochimique d'agents pathogènes dans l'air expiré Download PDF

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Publication number
WO2023192433A1
WO2023192433A1 PCT/US2023/016822 US2023016822W WO2023192433A1 WO 2023192433 A1 WO2023192433 A1 WO 2023192433A1 US 2023016822 W US2023016822 W US 2023016822W WO 2023192433 A1 WO2023192433 A1 WO 2023192433A1
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Prior art keywords
biosensor
ebc
virus
pathogens
exhaled breath
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PCT/US2023/016822
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English (en)
Inventor
Benjamin SUMLIN
Rajan CHAKRABARTY
Nishit Jaideep SHETTY
John CIRRITO
Carla YUEDE
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Washington University
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Publication of WO2023192433A1 publication Critical patent/WO2023192433A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
    • A61B5/097—Devices for facilitating collection of breath or for directing breath into or through measuring devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
    • A61B5/082—Evaluation by breath analysis, e.g. determination of the chemical composition of exhaled breath
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the field of the disclosure relates generally to devices, systems, and methods for rapidly and reliably collecting and analyzing exhaled breath, and real-time detection of pathogens in exhaled breath.
  • Coronavirus disease 2019 (COVID-19), first reported in December 2019, has swept the world, resulting in nearly 5.8 million deaths worldwide and more than 900,000 deaths in the United States as of early February 2022 (WHO website).
  • the disease is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; CoV-2) which is primarily transmitted human-to-human through respiratory droplets and aerosols.
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • COVID- 19 vaccines have been effective in reducing hospitalizations and severe illness among vulnerable populations; however, the vaccines do not prevent human-to-human virus transmission and have reduced efficacy against new variants, such as omicron and BA.2.
  • Current tests for CoV-2 vary in sensitivity, specificity, and test response times.
  • Quantitative RT-PCR tests to detect viral RNA vary in response time from hours to days, while antibody tests generally take at least 1-2 days for results. Antigen tests tend to be the fastest, with current saliva or nasal swab results returned in as little as 5 minutes (e.g., Abbott COVID- 19 ID NOW Test); however, these tests are invasive, have moderate specificity, and are not feasible to conduct rapidly on large groups of people. Moreover, the high rate of false positives with antigen tests can result in unnecessary and potentially hazardous therapeutic treatment of individuals who are not infected.
  • Rapid and real-time detection of the virus can help slow the spread by detecting the virus and allowing for quick disinfection, contact tracing and isolation.
  • rapid and real-time detection of the virus can help slow the spread by detecting the virus and allowing for quick disinfection, contact tracing and isolation.
  • the present disclosure is directed to an exhaled breath condensate (EBC) collection device for collecting pathogens for analysis, the device comprising: a breathing tube; and a condensing chamber located downstream from and in flow communication with the breathing tube, wherein the condensing chamber includes an upper chamber and a lower chamber separated by an inclined superhydrophobic surface.
  • the EBC collection device further comprises a biosensor.
  • the inclined superhydrophobic surface is inclined at about 10 degrees to about 45 degrees
  • the superhydrophobic impaction surface is a polyimide film
  • the polyimide film is coated with a spray-coating of silicone wax
  • the lower chamber contains a cooling fluid
  • the cooling fluid is water
  • the cooling fluid is produced from a user- initiated endothermic reaction.
  • the EBC device further comprises at least one transport fluid reservoir located upstream from and in flow communication with the condensing chamber.
  • the at least one transport fluid reservoir contains a buffer.
  • the buffer is phosphate-buffered saline.
  • the at least one transport fluid reservoir contains a blocking agent.
  • the blocking agent is bovine serum albumin.
  • the at least one transport fluid reservoir contains a combination of phosphate-buffered saline and bovine serum albumen.
  • the EBC device further comprises at least one disinfectant reservoir located upstream from and in flow communication with the condensing chamber.
  • the at least one disinfectant reservoir contains hypochlorous acid (HOC1).
  • the EBC device is disposable.
  • the present disclosure is directed to a method for detecting pathogens in exhaled breath, the method comprising: introducing an exhaled breath sample into an exhaled breath condensate (EBC) collection unit; condensing the exhaled breath sample into a liquid phase sample; contacting the liquid phase sample with a biosensor; measuring an output of the biosensor; and detecting at least one pathogen in the exhaled breath sample.
  • EBC exhaled breath condensate
  • the exhaled breath sample is condensed on a superhydrophobic impaction surface.
  • the method further comprises contacting the superhydrophobic impaction surface with a transport fluid to wash the condensed liquid phase sample onto the biosensor.
  • measuring the output of the biosensor comprises connecting a potentiostat unit to the biosensor and measuring a current output of the biosensor, wherein the current output of the biosensor is based on a square wave voltammetry measurement of tyrosine oxidation of tyrosine.
  • the method further comprises flushing the EBC with a disinfectant and disposing of the EBC unit.
  • the present disclosure is directed to a system for detecting pathogens, the system comprising: a disposable exhaled breath condensate (EBC) collection unit; and a portable potentiostat unit.
  • EBC disposable exhaled breath condensate
  • the portable PU comprises a potentiostat, a battery, a user interface, and a microcontroller, and the portable PU is reusable.
  • the system for detecting pathogens is used to detect viruses, bacteria, parasites, fungi, mold, or a combination thereof. In some embodiments, the system for detecting pathogens is used to detect multiple pathogens in a single test. In other embodiments, the system for detecting pathogens is used to detect multiple pathogens simultaneously in a single test, wherein the multiple pathogens are selected from combinations of viruses, bacteria, parasites, fungi, and mold, or multiple viruses, multiple bacteria, or multiple species or strains of viruses, bacteria, parasites, fungi, or mold. In some embodiments, the system for detecting pathogens is used to test for multiple variants of a pathogen (e.g., delta vs. omicron variants of SARS-CoV-2) from a single test. In other embodiments, the system for detecting pathogens is used in multiplex tests in which it detects multiple pathogens simultaneously.
  • a pathogen e.g., delta vs. omicron variants of
  • FIG. 1 is an exemplary embodiment of SARS-CoV-2 biosensor design in accordance with the present disclosure.
  • the CoV-2 biosensor uses square wave voltammetry to measure oxidation of tyrosine amino acids within the viral particle. Oxidation releases electrons that the sensor detects as current.
  • the biosensor uses a nanobody attached to the surface to provide specificity and concentrate the viral particle at the electrode for measurement. The electrode is blocked with albumin to limit non-specific signal.
  • FIGs. 3A-3D depict an exemplary embodiment of breathalyzer technology in accordance with the present disclosure.
  • FIG. 3A shows a separated schematic of the EBC device. A user breathes into the tube. On the inside is a thermoconductive film (orange) covered in a hydrophobic layer that is cooled to 10°C. The exhaled breath is condensed on the surface then washed onto the biosensor using a transport media to measure for one or more pathogens.
  • FIG. 3B shows a prototype design printed in resin.
  • FIG. 3C shows an annotated interconnection between the EBC device and a portable, battery-powered electrometer.
  • FIG. 3D shows a method for detecting pathogens in exhaled breath.
  • FIG. 4 is an exemplary embodiment of oxidation peak signals measured using the biosensor for varying concentrations of CoV-1 and CoV-2 spike protein on the same electrode in accordance with the present disclosure.
  • FIG. 5 is an exemplary embodiment of a dilution series of inactivated viral particles on the CoV-2 biosensor in accordance with the present disclosure. Chemically inactivated viral particles of the WA.l, beta, delta, and omicron (BA.l) strains were tested.
  • FIGs. 6A-6B depict an exemplary embodiment of aerosols with inactivated viral particles were nebulized, collected by the EBC, then detected on the biosensor in accordance with the present disclosure.
  • FIG. 6A shows an example of the size distribution of the aerosolized particles, with a peak around lOOnm in diameter.
  • FIGs. 7A-7D depict an exemplary embodiment of a central analysis unit in accordance with the present disclosure.
  • FIG. 7A shows a front of the device.
  • FIG. 7B shows an internal view of a microcontroller and peristaltic pumps.
  • FIG. 7C shows a rear of the device, which has fluid ports, power entry, and an optional networking port.
  • FIG. 7D shows an internal view, which has pumps, power supply, and a location for an electrode vial (not installed).
  • the methods and systems described herein embody low-cost, rapid detection techniques in a fast, disposable, single-use test envisioned for scalable production (e.g., for use in hospitals, airports, schools, military bases, military vessels, and anywhere where a large number of people are expected to gather).
  • Results of the rapid test methods and systems disclosed herein include real-time results as well as results in less than about 120s, about 90s, about 75s, about 60s, about 45s, about 30s, or about 15s.
  • sampling, sample processing, analyzing and/or detecting techniques may be carried out in a sterile environment (or environments), depending upon the embodiment.
  • the electrochemical methods and systems exemplify improved limits of detection and higher fidelity than current available “rapid” antigen tests.
  • a breathalyzer for real-time detection of aerosolized pathogens (including SARS-CoV-2) using an immuno-based biosensor.
  • the breathalyzer is primarily for rapid testing of infected individuals (both symptomatic and asymptomatic) at a diagnostic level within 60 seconds of breathing into the device with the expectation that follow-up testing is not required.
  • a single-use disposable exhaled breath condensate (EBC) collection device has been developed for capturing breath aerosols. The patient blows into the collection device which consists of a cooled (-10° C) hydrophobic film on the interior. The temperature difference between the exhaled breath and cool surface leads to condensation-based growth and collection of aerosol particles.
  • Test buffer is added to collect the breath condensate which is applied to the biosensor for detection.
  • the breath condensate is applied to the biosensor and subsequently analyzed within several seconds or minutes of addition of the test buffer.
  • an exhaled breath sample may be collected (e.g., condensed and washed with test buffer) and suitably stored for a period of time (e.g., hours, days, weeks, etc.) prior to contact with the biosensor for pathogen detection.
  • a period of time e.g., hours, days, weeks, etc.
  • remote or at-home testing EBC collection devices may be provided such that collected samples require storage and/or transportation prior to contact with a biosensor for analysis.
  • the breathalyzer is deployable to hospitals, schools, airports, and military facilities/vessels where a long queue of individuals needs to be rapidly tested. These devices will provide rapid readouts and act as a platform to detect other respiratory viruses and emerging pathogens.
  • Limit of detection (LoD) of the biosensor The biosensors were tested with varying concentrations of chemically-inactivated SARS-CoV-2 viral particles. WA.l, beta, delta, and omicron BA.1 strains were tested with high sensitivities on the biosensor at 10-50 viral particles/virions/RNA copies per sample as verified by qRT-PCR. In some embodiments, the devices and systems disclosed herein have a limit of detection of less than 10 viral particles.
  • the biosensor nanobody detects the repeat binding domain of the SARS-CoV-2 spike protein.
  • the biosensor was tested against recombinant SARS-CoV-2 compared to a comparable protein sequence in SARS-CoV-1.
  • CoV-1 produced negligible signal on the biosensor with over a 1,000-fold selectivity for CoV-2 over CoV-1 spike protein.
  • the biosensor detects inactivated viral particles of at least one respiratory virus (or several respiratory viruses) alternatively or additional to SARS-CoV-2 virus particles.
  • the biosensor detects at least one bacterial genus or species.
  • the device detects at least one parasite, fungi, or mold genus or species.
  • Viral particles detectable by the disclosed biosensor include, but are not limited to, viruses associated with Chikungunya, Cholera, Crimean-Congo hemorrhagic fever, Ebola virus disease, Hendra virus infection, Influenza (pandemic, seasonal, zoonotic), Lassa fever, Marburg virus disease, Meningitis, MERS-CoV, Monkeypox, Nipah virus infection, Novel coronavirus (2019-nCoV), Plague, Rift Valley fever, SARS, Smallpox, Tularaemia, Yellow fever, Zika virus disease, Ebola and Marburg virus (Filoviridae); Ross River virus, chikungunya virus, Sindbis virus, eastern equine encephalitis virus (Togaviridae, Alphavirus), vesicular stomatitis virus (Rhabdoviridae, Vesiculovirus), Amapari virus, Pichinde virus, Tacaribe virus, Junin virus, Machupo virus
  • louis encephalitis vims Flavivims, Flaviviridae Tick-borne powassan vims Flavivims, Flaviviridae Torque teno vims Alphatorquevims, Anelloviridae Toscana vims Phlebovims, Bunyaviridae Uukuniemi vims Phlebovims, Bunyaviridae Vaccinia vims Orthopoxvirus, Poxviridae Varicella-zoster vims Varicellovims, Herpesviridae Variola vims Orthopoxvirus, Poxviridae Venezuelan equine encephalitis Alphavims, Togaviridae vims Vesicular stomatitis vims Vesiculovims, Rhabdoviridae Western equine encephalitis vims Alphavims, Togaviridae WU polyomavims, Polyomavirid
  • Bacterial genera and species detectable by the disclosed biosensor include, but are not limited to, bacteria associated with Xanthomonas, Pseudomonas, Salmonella, Shigella, Chlamydia, Helicobacter, Yersinia, Bordatella, Pseudomonas, Neisseria, Vibrio, Haemophilus, Mycoplasma, Streptomyces, Treponema, Coxiella, Ehrlichia, Brucella, Streptobacillus, Fusospirogna, Spirillum, Ureaplasma, Spirochaeta, Mycoplasma, Actinomycetes, Borrelia, Bacteroides, Trichomoras, Branhamella, Pasteurella, Clostridium, Corynebacterium, Listeria, Bacillus, Erysipelothrix, Rhodococcus, Escherichia, Klebsiella, Pseudomanas, Entero
  • E. coli P. cepacia
  • S. epidermis E. faecalis
  • S. pneumonias S. aureus
  • N meningitidis S. pyogenes
  • Pasteurella multocida Treponema pallidum, and P. mirabilis.
  • Gram-negative bacterial genera and species detectable by the disclosed biosensor include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp.
  • Gramnegative bacterial genera and species detectable by the disclosed biosensor include, but are not limited to, Salmonella, E. coli, Yersinia pestis, Klebsiella and Shigella, Proteus, Enterobacter, Serratia, and Citrobacter.
  • Fungi detectable by the disclosed biosensor include, but are not limited to, fungi associated with Cryptococcus neoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis; Histoplasma capsulatum; Coccidioides immitis; Candida species, including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii and C. krusei, Aspergillus species, including A. fumigatus, A. flavus and A. niger, Rhizopus species; Rhizomucor species; Cunninghammella species; Apophysomyces species, including A. saksenaea, A.
  • Parasites detectable by the disclosed biosensor include, but are not limited to, parasites associated with Anaplocephala, Ancylostoma, Necator, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Cyathostomum, Cylicocyclus, Cylicodontophorus, Cylicostephanus, Craterostomum, Dictyocaulus, Dipetalonema, Dipylidium, Dracunculus, Echinococcus, Enterobius, Fasciola, Filaroides, Habronema, Haemonchus, Metastrongylus, Moniezia, Nematodirus, Nippostrongylus, Oesophagostomum, Onchocerca, Ostertagia, Oxyuris, Parascaris, Schistosoma, Strongylus, Taenia, Toxocara, Strongyloides, Toxascaris, Trichinella, Trich
  • Additional pathogens detectable by the disclosed biosensor include, but are not limited to, Coronaviridae (e.g. MERS, SARS-CoV-2), Bunyavirales (e.g. Lassa, Junin, Rift Valley Fever Virus, Andes, Sin Nombre, LaCrosse, California Encephalitis, Crimean Congo Hemorrhagic Fever), Filoviruses (e.g. Ebola, Marburg), Flaviviruses (e.g. Dengue, Zika, West Nile), Paramyxoviridae (e.g. Nipah, Hendra), Picomaviridae (e.g. EV-D68, EV- A71), Togaviridae (e.g.
  • Coronaviridae e.g. MERS, SARS-CoV-2
  • Bunyavirales e.g. Lassa, Junin, Rift Valley Fever Virus, Andes, Sin Nombre, LaCrosse, California Encephalitis
  • Chikungunya EEE, VEE, WEE
  • Bacillus anthracis including genotypic resistance markers
  • Yersinia pestis including genotypic resistance markers
  • Francisellatularensis including genotypic resistance markers
  • genotypic resistance markers e.g., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp
  • Lassa virus Nipah virus, Rift Valley Fever virus, Enterovirus D68 virus, Candida auris, Coccidioides sp., and novel coronaviruses.
  • An exhaled breath condensate (EBC) collection device for collecting pathogens for analysis, the device comprising: a breathing tube; and a condensing chamber located downstream from and in flow communication with the breathing tube, wherein the condensing chamber includes an upper chamber and a lower chamber separated by an inclined superhydrophobic impaction surface.
  • EBC exhaled breath condensate
  • the EBC collection device of any preceding clause further comprising at least one transport fluid reservoir located upstream from and in flow communication with the condensing chamber.
  • the at least one buffer includes a phosphate buffer, a tris buffer, or a saline buffer.
  • the at least one transport fluid reservoir comprises a first transport fluid reservoir containing phosphate-buffered saline and a second transport fluid reservoir containing bovine serum albumen.
  • a method for detecting pathogens in exhaled breath comprising: introducing an exhaled breath sample into an exhaled breath condensate (EBC) collection unit; condensing the exhaled breath sample into a liquid phase sample; contacting the liquid phase sample with a biosensor; measuring an output of the biosensor; and detecting at least one pathogen in the exhaled breath sample.
  • EBC exhaled breath condensate
  • measuring the output of the biosensor comprises connecting a potentiostat unit to the biosensor and measuring a current output of the biosensor.
  • a system for detecting pathogens comprising: a disposable exhaled breath condensate (EBC) collection unit; and a portable potentiostat unit.
  • EBC exhaled breath condensate
  • Particle collection efficiency The current EBC particle collection efficiency for the breathalyzer is 47.2 ⁇ 19.2%. This efficiency value was determined with aerosols nebulized at room temperature; however, aerosols in breath typically have a higher temperature and condense more readily on a cool surface which will increase collection efficiency. In some embodiments, the breathalyzer device has an aerosol collection efficiency above 70%.
  • the aerosol collection devices and the biosensor tests include those for crossreactivity to other respiratory viruses and endogenous/exogenous substances found in breath as indicated by FDA guidance for the breathalyzer.
  • the disclosed solution entails the manufacture of a breathalyzer for point-of-care diagnosis of both symptomatic and asymptomatic pathogen detection (e.g., SARS-CoV-2).
  • SARS-CoV-2 a breathalyzer for point-of-care diagnosis of both symptomatic and asymptomatic pathogen detection
  • CHeST is an aerosol generation device which produces conditions and expelled particles analogous to those observed during respiratory exhalation and coughing. The length and interval of aerosol generation is user-controlled to mimic breathing patterns of individuals with different lung capacities and expiratory flow rates.
  • a nanobody-based electrochemical biosensor is used to selectively detect CoV-2 virions via the spike protein with a high sensitivity as low as 10-50 viral parti cles/sample.
  • the technique provides instant results — within 60 seconds for the breathalyzer (for instance: 30 seconds to collect EBC, 15 seconds transfer to biosensor, 15 seconds to measure).
  • the speed and sensitivity of this test enable improved prevention of indoor space disease transmission while simultaneously facilitating more indoor activity with a higher level of safety.
  • the described technology has been successful in detecting prevalent SARS- CoV-2 variants such as the WAI, B.1.351 (beta), B.1.617.2 (delta), and BA. l (omicron) strains.
  • the technology is a platform that is modifiable to detect other non- COVID pathogens such as influenza, OC43, MERS, and biological weapons such as anthrax.
  • the breathalyzer device is herein enabled as a stand-alone instrument to be deployed to multiple locations and operated and maintained by technicians with minimal training.
  • Breathalyzer device testing according to the present disclosure involves using a device to mimic various types of expiration, including regular breaths, shallow breaths, talking, sneezing and coughing. As described herein, testing and optimization of each of the breathalyzer, environmental detector, and the biosensor includes interference from other factors, such as other respiratory viruses and endogenous/exogenous compounds in breath, as well as environmental pollutants.
  • an electrochemical nanobody-based biosensor has been developed to detect aerosolized CoV-2 that will be deployed in a breathalyzer for real-time diagnosis.
  • the environmental biosensor of the present disclosure for detecting target organisms is surprisingly and unexpectedly based on an ultra-sensitive electrochemical technology used in vivo (e.g., brain, tissue, interstitial fluid, etc.) for Alzheimer’s disease research for detecting macromolecular targets.
  • the breathalyzer is deployable to testing locations that require rapid testing of a queue, such as lines to enter an immigration hall, a school, hospital, airplane, military vessel, or entertainment venue.
  • the breathalyzer device comprises a single-use disposable EBC collector (a cm3 box that the user blows into) that contains a tube for breathing into, a cold hydrophobic surface to condense the aerosols, separate chambers to hold wash buffer and HOC1 disinfection solution, and the SARS-CoV-2 biosensor.
  • the disposable EBC collector is then connected to a stationary, permanent analysis unit that contains the potentiostat needed to control the biosensor and a computer to analyze the signal to generate a diagnosis of positive or negative SARS-CoV-2 status with a simple user interface.
  • the EBC collector is internally disinfected with HOC1 then discarded after a single use to prevent infection of the operator.
  • the nanobody/biosensor can be implemented with new variants requiring screening and selection of a new nanobody.
  • the nanobody/biosensor demonstrated herein detects the WA. l, alpha, beta, delta, and omicron BA. l strains at 50 virions/ml or lower, which meets pre-determined parameters.
  • the present disclosure enables re-testing of existing nanobody libraries and/or development of a new nanobody (or antibodies).
  • the disclosed breathalyzer device can improve national testing capacity as it is a fast and non-invasive technique to test queues of people waiting to enter indoor spaces, such as airports, hospitals, conference centers, or military installations, etc., where virus transmission may be prevalent.
  • the innovations of the present disclosure include an immuno-based biosensor with a nanobody to provide specificity, as well as collection components, devices, and systems to collect aerosolized pathogens (e.g., CoV-2 viral particles) prior to testing on the biosensor.
  • Nanobody-based electrochemical biosensor for real-time detection of aerosolized pathogens [0080] Nanobody-based electrochemical biosensor for real-time detection of aerosolized pathogens
  • the present disclosure describes a novel single- step method to sample exhaled breath, condense it to liquid phase and deliver it to a screen- printed electrode (SPE) that has been specially prepared for the electrochemical detection of pathogens (such as SARS-CoV-2 and others, depending on the preparation method embodiment).
  • SPE screen- printed electrode
  • a system embodiment of the disclosure comprises at least two units - (the EBC collection unit), and a portable potentiostat unit (PU).
  • the EBC is comprised of an inclined (from about 10 degrees to about 45 degrees) superhydrophobic impaction surface, optionally the SPE, optionally a cold fluid to lower the temperature of the impaction surface, optionally at least one reservoir containing extra working fluid (or transport fluid), optionally a disinfectant, and a breathing tube.
  • the EBC includes the inclined superhydrophobic impaction surface, an electrode, a first reservoir containing a buffer, a second reservoir containing a blocking agent, and a third reservoir containing a disinfectant.
  • the superhydrophobic impaction surface may be any suitable hydrophobic surface, including but not limited to, a film with an optional coating such as a spray coating.
  • the buffer may be any suitable buffer, including but not limited to, a phosphate buffer, a tris buffer, or a saline buffer.
  • the blocking agent may be any suitable blocking agent, including but not limited to, a plant or animal based albumen.
  • the disinfectant may be any suitable disinfectant, including but not limited to, hypochlorous acid (HOC1).
  • the EBC includes a superhydrophobic impaction surface made of polyimide treated with a spray-coating of silicone wax, a screen-printed carbon electrode, a first reservoir containing phosphate-buffered saline, a second reservoir containing bovine serum albumen, and a third reservoir containing a hypochlorous acid (HOC1) disinfectant.
  • the PU includes a potentiostat, a battery, a user interface, and a microcontroller.
  • the EBC includes a condensing chamber separated into two chambers (upper and lower) separated by the polyimide film.
  • a cold fluid such as ice water or any suitable cold fluid
  • the temperature of the cold fluid, the lower chamber, and/or the superhydrophobic surface is maintained from about 0°C to about 10°C or from about 5°C to about 15°C, or from about 10°C to about 20°C, or from about 15°C to about 25°C.
  • the gas and droplet mixture impacts the polyimide, condenses into liquid, and rolls off the inclined superhydrophobic surface into a small vial containing the SPE.
  • Extra working fluid (or transport fluid) is released to wash all collected breath onto the SPE.
  • the SPE’s exposed contacts are then plugged into the PU unit, a reading is conducted, and the results are reported.
  • the PU is disconnected and the EBC is flushed with HOC1 to sterilize it.
  • the sterilized enclosure can then be disposed of without any risk of pathogen leak/transfer to the environment.
  • Electrochemical biosensor Described herein is a micro-immunoelectrode (MIE) technology that uses square wave voltammetry to measure oxidation of tyrosine amino acids (at -0.65 V) in specific proteins (i.e., pathogen-indicating proteins). Tyrosine oxidation releases electrons that a carbon electrode detects as current (FIG. 1). The amount of current is directly proportional to the amount of analyte present.
  • An antibody covalently attached to the electrode surface concentrates the target at the biosensor for measurement.
  • a nanobody produced in llamas then sequenced and grown cost effectively in bacteria
  • one or more nanobodies are attached to the biosensor, alternative or additional to a SARS-CoV-2 nanobody.
  • a signal e.g., current
  • tyrosine oxidation is irreversible, meaning the protein bound to the nanobody on the surface of the electrode will only be measured once. This contrasts with many electrochemical sensors that measure impedance at the electrode surface; essentially measuring the binding event instead of the actual protein.
  • the disclosed biosensor uses screen-printed, inexpensive, carbon-based electrodes (SPiCE).
  • MIE micro-immunoelectrode
  • the A0 biosensors are surgically implanted into the mouse brain, enabling real-time measurement of the brain interstitial fluid in mice that are awake and freely moving. While the A0 and SARS-CoV-2 designs are different (5pm carbon fiber pulled in glass versus a 1mm screen printed electrode, respectively) based on their intended uses, the principle underlying the biosensors is analogous.
  • the A0 biosensor was implanted into the brains of 1) APP/PS1 transgenic mice that express human A0 or 2) wild-type mice that only express endogenous murine A0.
  • murine A0 lacks the tyrosine amino acid in human A0 that, according to the theory of how the biosensors work, is required to produce the electrochemical signal on the biosensor, serving as a powerful control for specificity in vivo.
  • the biosensor measured human A0 every 60 seconds for 3 hours with minute-to-minute variability that is expected based on on-going neuronal activity (FIG. 2).
  • signal in the wild-type mice was negligible for the entire 3 -hour measurement period.
  • the A0 biosensor is 8,000-fold more selective for human A0 than any other tyrosine in the brain.
  • a series of biosensors were developed for use in a variety of mouse models of neurological disease, including targeting various species of A0 peptide (A04o, A042, and A0 oligomers), tau, and a-synuclein.
  • A0 peptide A04o, A042, and A0 oligomers
  • tau tau
  • a-synuclein Another MIE was also developed against met- enkephalin, a neuromodulator peptide.
  • Standard A0 oligomer ELIS As are generally sensitive to the low pg/ml range, whereas the biosensor is sensitive to 200 attogram/ml levels of oligomers, an approximate 10,000-fold increase in sensitivity.
  • SARS-CoV-2 breathalyzer A single-use technology was developed to sample exhaled breath, condense it to liquid phase, and deliver it to the biosensor (FIG. 3(A- D)).
  • the inside of the device includes a chilled superhydrophobic surface, such that when a warm breath contacts the cold surface, the difference in temperature causes the aerosol particles to condense on the surface.
  • the hydrophobic layer is currently chilled with water, however the final device will be chilled by a user-initiated endothermic reaction (similar to a chemical ice pack).
  • a user blows into the collector through a breathing tube or straw. The blowing process leads to impaction of particles on the chilled film which condenses the aerosol particles.
  • a transport media washes the aerosol droplets into a liquid phase sample for measurement upon contact with a biosensor.
  • the biosensor may be a single-use biosensor that is integrated into the singleuse EBC collection device, or alternatively the biosensor may be a reusable (i.e., multi-use) biosensor that is integrated into an external analysis unit.
  • the aerosol droplets are washed onto the biosensor and the biosensor is then plugged into an external analysis unit/docking station that houses the potentiostat and software to control the biosensor and measure for SARS-CoV-2.
  • the liquid phase sample is suitably contacted with the biosensor and measured.
  • a disinfectant such as hypochlorous acid (H0C1) or any other suitable disinfectant is released into the EBC collection unit to disinfect the device.
  • H0C1 hypochlorous acid
  • the entire collection device is designed to be disposable and self-disinfecting, while the analysis unit will be permanent and reusable.
  • FIG. 3A illustrates an exemplary EBC collection unit 300.
  • Exhaled breath is introduced (e.g., by breathing or blowing) into EBC unit 300 via breathing tube 302.
  • EBC unit 300 also includes a condensing chamber 304 located downstream from and in flow communication with breathing tube 302.
  • Condensing chamber 304 includes an upper chamber (not shown) and a lower chamber 306 separated by a superhydrophobic surface 308.
  • the upper chamber is generally defined by the space above surface 308 within condensing chamber 304.
  • Lower chamber 306 holds a cooling fluid capable of maintaining surface 308 at a temperature conducive to condensation of exhaled breath from aerosol particles into a liquid phase 310.
  • the cooling fluid is water or a fluid produced from a user-initiated (i.e., at or immediately before the time of exhaled breath collection) endothermic reaction.
  • surface 308 is a superhydrophobic impaction surface positioned at an incline of about 10 degrees to about 45 degrees.
  • Surface 308 may be a suitable thermoconductive film such as polyimide, with a suitable superhydrophobic coating such as a spray-coating of silicone wax.
  • EBC unit 300 further includes a biosensor 312 located below/downstream from surface 308 such that condensed liquid phase 310 flows down onto biosensor 312.
  • Biosensor 312 includes exposed contact(s) 314 which extend out of EBC unit 300 for connection/attachment (e.g., see FIG. 3C) to a portable potentiostat unit, a portable battery-powered electrometer , and/or an analysis unit as described herein elsewhere.
  • EBC unit 300 optionally includes additional reservoirs (not shown) located upstream from condensing chamber 304 for introducing transport fluid for washing surface 308 and/or for introducing disinfectant for selfdisinfection of EBC unit 300 prior to disposal (such as for single-use units).
  • Transport fluid also termed working fluid or test buffer
  • a buffer such as phosphate buffered saline
  • a blocking agent such as bovine serum albumen
  • Disinfectant includes hydrochlorous acid (HOC1) as well as other suitable disinfectants.
  • transport fluid and/or disinfectant fluid is introduced into condensing chamber 304 via port 316 (see FIG. 3B).
  • FIG. 3C shows EBC unit 300 with biosensor 312 communicatively coupled to a portable electrometer 318, e.g., via contact(s) located on biosensor 312 (such as exposed contacts 314 shown in FIG. 3 A).
  • portable electrometer 318 may be battery powered and/or may be additionally or alternatively configured as a portable potentiostat unit.
  • biosensor 312 may be communicatively coupled to a non-portable type analysis unit.
  • FIG. 3D further shows an exemplary method 320 for detecting pathogens in exhaled breath according to the present disclosure.
  • Method 320 includes introducing 322 an exhaled breath sample into an EBC collection unit, condensing 324 the exhaled breath sample into a liquid phase sample, contacting 326 the liquid phase sample with a biosensor; measuring 328 an output of the biosensor; and detecting 330 at least one pathogen in the exhaled breath sample.
  • the exhaled breath sample is condensed on a superhydrophobic impaction surface.
  • exemplary embodiments of the present disclosure include a system for detecting pathogens, the system comprising a disposable EBC collection unit and a potentiostat unit (PU).
  • the PU comprises, a battery, a user interface, and a microcontroller, and is reusable.
  • the method also comprises contacting the superhydrophobic impaction surface with a transport fluid to wash the condensed liquid phase sample onto the biosensor and measuring the output of the biosensor comprises connecting a potentiostat unit to the biosensor and measuring a current output of the biosensor, wherein the current output of the biosensor is based on a square wave voltammetry measurement of tyrosine oxidation of tyrosine.
  • the method further comprises flushing the EBC with a disinfectant and disposing of the EBC unit (such as for single-use units).
  • Biosensor specificity The specificity of the CoV-2 biosensor has been tested with CoV-1 and CoV-2 spike protein with CoV-2 being detected at the low pg/mL range and negligible signal for CoV-1 (FIG. 4).
  • the specificity of the final product is to be determined by comparing to a variety of inactivated viral particles, such as HCoV229E, OC43, HKU1, NL63, MERS, etc. Analysis shown here is supportive such that the device is expected to have each of sensitivity and specificity greater than 95%.
  • Biosensor sensitivity The biosensor was tested with a dilution series of different variants of inactivated SARS-CoV-2 (WA. l, beta, delta, and omicron BA. l) (FIG. 5). The biosensor was most sensitive for the delta and beta strains down to 10 RNA copies/ml. For all studies during development, the viral RNA copy numbers are verified using qRT-PCR. While early studies with the omicron variant demonstrate a lower limit sensitivity of 50 RNA particles/ml, a minor loss of sensitivity relative to the other variants, this is still well within the range necessary for human diagnosis. The initial tests described herein with omicron enable biosensor optimization for increased variant sensitivity. The current biosensors saturate and demonstrate a hook dose effect at high concentrations of viral particles. The length of the plateau in relation to the hook effect is still being determined for the various strains of virus.
  • Inactivated CoV-2 particles were aerosolized then collected using the EBC collection device.
  • the aerosolized particles had a mean peak of approximately lOOnm in diameter (FIG. 6A).
  • the collected aerosols were manually pipetted onto the biosensor for detection.
  • WA. l, delta, and omicron viral particles were readily detected on the biosensor compared to aerosolized control solution lacking virus (FIG. 6B).
  • FIGs. 7A-7D show a central analysis unit containing the electrochemical sensor along with all the working fluids.
  • This central analysis unit may be used by the environmental detector, and a similar design without the internal fluids may be used for the breathalyzer.
  • the central analysis unit for the environmental detector has been designed such that the entire process of sample transport and analysis can be automated. This device may be user friendly such that it can be deployed to multiple locations and be operated with minimal training.
  • numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.”
  • the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that vary depending upon the desired properties sought to be obtained by a particular embodiment.
  • the numerical parameters are be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) are construed to cover both the singular and the plural, unless specifically noted otherwise.
  • the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or to refer to the alternatives that are mutually exclusive.
  • compositions and/or methods disclosed and claimed herein may be made and/or executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of the embodiments included herein, it will be apparent to those of ordinary skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as defined by the appended claims.

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Abstract

La présente invention concerne un dispositif de collecte de condensat d'air exhalé (EBC) servant à collecter des agents pathogènes à des fins d'analyse, ledit dispositif comprenant : un tube respiratoire; et une chambre de condensation située en aval de et en communication fluidique avec le tube respiratoire, la chambre de condensation comprenant une chambre supérieure et une chambre inférieure séparées par une surface superhydrophobe.
PCT/US2023/016822 2022-04-01 2023-03-30 Procédés et systèmes rapides à usage unique pour l'analyse électrochimique d'agents pathogènes dans l'air expiré WO2023192433A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200016354A1 (en) * 2018-07-11 2020-01-16 Drägerwerk AG & Co. KGaA Ventilation system with electrochemical filter for alkyl phenols and method using the electrochemical filter
US20200397340A1 (en) * 2019-02-11 2020-12-24 Giner, Inc. Method and system for detection and/or quantification of delta-9-tetrahydrocannabinol in exhaled breath
US20210022673A1 (en) * 2018-07-31 2021-01-28 University Of North Texas Techniques for rapid detection and quantitation of volatile organic compounds (vocs) using breath samples
WO2021226406A1 (fr) * 2020-05-06 2021-11-11 Jerry Aguren Procédé et appareil photonique pour détecter des composés et des pathogènes dans un échantillon respiratoire
KR20220024190A (ko) * 2019-06-19 2022-03-03 엑설레이션 테크놀로지 리미티드 호기 호흡을 위한 수집 디바이스

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200016354A1 (en) * 2018-07-11 2020-01-16 Drägerwerk AG & Co. KGaA Ventilation system with electrochemical filter for alkyl phenols and method using the electrochemical filter
US20210022673A1 (en) * 2018-07-31 2021-01-28 University Of North Texas Techniques for rapid detection and quantitation of volatile organic compounds (vocs) using breath samples
US20200397340A1 (en) * 2019-02-11 2020-12-24 Giner, Inc. Method and system for detection and/or quantification of delta-9-tetrahydrocannabinol in exhaled breath
KR20220024190A (ko) * 2019-06-19 2022-03-03 엑설레이션 테크놀로지 리미티드 호기 호흡을 위한 수집 디바이스
WO2021226406A1 (fr) * 2020-05-06 2021-11-11 Jerry Aguren Procédé et appareil photonique pour détecter des composés et des pathogènes dans un échantillon respiratoire

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